HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

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Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript. The source codes for the whole blood RNA-Seq analysis (Study 1) and CD14+ cells RNA-seq analysis (Study 2) are available at: https://github.com/sekalylab/vb013. The source codes for the microRNA-Seq analysis (Study 1) are available at: https://github.com/NIDAP-Community/HIV-vaccine-candidate-efficacy. The source codes for the CD14+ cells RNA-Seq/ATAC-Seq analysis (Study 2) are available at: https://github.com/CBIIT/BM-Rhesus-NatureComm2023.git.
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Research was not conducted in human participants
Research was not conducted in human participants

Research was not conducted in human participants
Research was not conducted in human participants nature portfolio | reporting summary

March 2021
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. The samples size of each group was determined by consultation with our statistician. The studies were not powered to compare SIVmac251 viral acquisition following viral exposure between vaccinated groups but rather between vaccinated and control groups.
In Study 1 the power of the study was calculated based on previous results, in which animals vaccinated with DNA/DNA/ALVAC+gp120/ALVAC +gp120 showed a 52% vaccine efficacy (Vaccari et al., Nat Med, 2018). In young animals, the power of the study using 13 vaccinated and 27 control macaques and assuming 52% vaccine efficacy was 59%; whereas, in old animals, the power of the study using 17 vaccinated and 11 control macaques and assuming 52% vaccine efficacy was 29% In study 2 the power of the study was calculated based on previous results (Vaccari et al., Nat Med, 2018 and young of study 2) and was estimated as 81% when using 12 vaccinated and 37 control macaques. Sample size was determined also based on our previous nonhuman primate studies in which, in the majority of the cases, a n=12 in vaccinated groups allowed us to identify a significant reduction of risk of acquisition. Based on statistician consultation, it was established that the comparisons of other outcomes most likely had adequate power, but power analyses were not performed for them.
MicroRNA samples that showed significantly different read distribution compared to the other samples and a poor gene coverage were removed (T112_wk13 and H24G_wk13 in study 1) ATAC-seq samples with Fraction Reads in called peak regions (FRIP) <5% were removed (BM266 in study 2). Remaining samples had all FRIP>10% The nature of the samples analyzed in the present studies, the limited amount of each sample collected from each animal and the cost of the non-human primate studies do not allow us to replicate the experiments. In the reported assays the replicates are represented by each animal enrolled in the study. All the data have been obtained with validated assays that have been used in previous publish work.
Study 1: macaques were assigned to four groups based on their major histocompatibility status, age, and weight. One MamuA01+ and one MamuB17+ animal were included in the vaccinated group of the young cohort, whereas two MamuA01+ animals were included in the vaccinated group of the old cohort.
Study 2: the study included only 1 group and therefore the animals were not randomized. The study included one MamuA01+ and one MamuB17+ animal.
For macaques studies (immunizations and viral exposures): the investigators that prepared the reagents were unblinded whereas the veterinarian staff was blinded. For immunological assays: during the execution and analyses of the experiments the investigators were blinded. Investigators were unblinded after generating the results in order to perform the final analyses of the data and correlation studies. Note that full information on the approval of the study protocol must also be provided in the manuscript.
As reported in the Method section: To identify monocytic myeloid cells in Study 1 the following antibodies were used: PE-Cy7 anti-CD3 (clone SP34-2; cat. #563916, 2. The study did not involve wild animals All animals used in these studies were female. The aim included evaluating the vaccine efficacy following vaginal exposure, therefore the sex of the animals was female The study did not involve samples collected from the field All animals were handled in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) in an AAALAC-accredited facility (OLAW, Animal Welfare Assurance A4149-01 for NIH and A3086-01 for Bioqual). All animal care and procedures were carried out under protocols approved by the NCI and/or NIAID Animal Care and Use Committees (ACUC; Protocol numbers: VB-013 and VB-026 at the NIH and P-181 at Bioqual).

March 2021
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Methodology
Sample preparation Instrument Software Cell population abundance Gating strategy Tick this box to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
Flow cytometry acquisitions for Study 1 were performed on on an an LSRII and examined using FACSDiva software (BD Biosciences) by by acquiring a minimum of of 500,000 events for myeloid cell evaluation. Flow cytometry acquisitions for Study 2 were performed on on a FACSymphony A5 A5 and examined using FACSDiva software (BD Biosciences) by by acquiring all stained cells.
FACSDiva software (BD Biosciences) and FlowJo v10.1 (TreeStar, Inc., Ashland, OR) For the identification of of monocytic myeloid cells the analyses were conducted on on Ficoll isolated PBMCs.
For ATAC-seq and RNA-seq in in CD14+ cells of of Study 2 the purity of of CD14+ cells was assessed by by flow cytometry. Following isolation, the purity was assessed using the following antibodies: PE-Cy7 anti-CD20 (clone 2H7; cat. #560735, 2.0 µl), Monocyte populations were identified as as CD3-CD20-CD45+HLA-DR+ and differentiated by by the expression of of CD14 and CD16, as as previously published. Classical monocytes were identified as as CD14+CD16-, Intermediate as as CD14+CD16+, and Non-Classical as as CD14-CD16+. Monocyte subsets were expressed either as as frequency of of the parental HLA-DR+ gate or or the live cells. The expression of of CCR2 in in each monocytic subset was expressed as as the frequency of of CCR2+ cells in in their parental subset. DC-10 cells were identified as as either Myeloid cells (high SSC)/Singlets/Live CD45+/CD3-CD20-/HLA-DR+/ CD1c-/CD11b+/CD11c+/ CD14+CD16+/CD163+/CD141+/CD1a-) and expressed as as the frequency of of the cells in in the final gate (CD1a-) of of the cells in in CD11b+ gate. Boundaries between positive and negative cells were established by by the neat separation of of the 2 major populations when the abundance of of the cells was allowing the distinction, or or by by the use of of FMO (Fluorescence minus one) when the cellular marker was had low expression or or the positive populations were rare A figure exemplifying the gating strategy is is provided in in the Supplementary Information only for Efferocytosis assay and DC-10 evaluation conducted in in Study 2